Persistent immune responses in the heart determine the outcome of cardiomyopathy syndrome in Atlantic salmon (Salmo salar).

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) is a severe cardiac disease in Atlantic salmon (Salmo salar) and one of the leading causes of morbidity and mortality in the Norwegian aquaculture industry. Previous research suggest a variation in individual susceptibility to develop severe disease, however the role of the immune response in determining individual outcome of CMS is poorly understood particularly in cases where fish are also challenged by stress. The present study's aim was therefore to characterize cardiac transcriptional responses to PMCV infection in Atlantic salmon responding to infection under stressful conditions with a high versus low degree of histopathological damage. The study was performed as a large-scale controlled experiment of Atlantic salmon smolts from pre-challenge to 12 weeks post infection (wpi) with PMCV, during which fish were exposed to intermittent stressors. RNA sequencing (RNAseq) was used to compare the heart transcriptome of high responders (HR) with atrium histopathology score '4' and low responders (LR) with score '0.5' at 12 wpi. A high-throughput quantitative PCR (qPCR) analysis was used to compare immune gene transcription between individuals sampled at 6, 9 and 12 wpi. Based on RNAseq and qPCR results, RNAscope in situ hybridization (ISH) was performed for visualization of IFN-γ - and IFNb producing immune cells in affected heart tissue. Compared to LR, the transcription of 1592 genes was increased in HR at 12 wpi. Of these genes, around. 40 % were immune-related, including various chemokines, key antiviral response molecules, and genes. associated with a Th1 pro-inflammatory immune response. Further, the qPCR analysis confirmed. increased immune gene transcription in HR at both 9 and 12 wpi, despite a decrease in PMCV. transcription between these time points. Interestingly, increased IFNb transcription in HR suggests the. presence of high-quantity IFN secreting cells in the hearts of these individuals. Indeed, RNAscope. confirmed the presence of IFN-γ and IFNb-positive cells in the heart ventricle of HR but not LR. To conclude, our data indicate that in severe outcomes of PMCV infection various chemokines attract leucocytes to the salmon heart, including IFN-γ and IFNb-secreting cells, and that these cells play important roles in maintaining persistent antiviral responses and a sustained host immunopathology despite decreasing heart viral transcription.

Complete genome sequence of vaccinium-associated virus C, a new member of the family Totiviridae from Vaccinium floribundum.

Blueberries (Vaccinium sp.) are a major crop grown in the Pacific Northwest region. Currently, there are at least 17 known viruses that infect blueberry plants, and some of them cause a wide range of symptoms and economic losses. A new virus, vaccinium-associated virus C (VaVC) (family Totiviridae, genus Totivirus) was identified in an imported blueberry accession from the USDA-ARS National Clonal Germplasm Repository in Corvallis, Oregon. The complete genomic sequence of VaVC was determined, but the biological significance of VaVC is unknown and requires further study. Additional Vaccinium sp. accessions should be screened to investigate the incidence of this new virus.

Molecular characterization of birch toti-like virus, a plant-associated member of the new family Orthototiviridae.

A novel totivirus, named "birch toti-like virus" (BTLV), was discovered in European white birch (Betula pendula) plants. The genome of BTLV is 4,967 nucleotides long and contains two overlapping open reading frames (ORFs) coding for the capsid protein (CP) and an RNA-dependent RNA-polymerase (RdRP). The encoded CP and RdRP proteins shared 46.9% and 60.2% amino acid sequence identity, respectively, with those of Panax notoginseng virus B. The presence of a putative slippery heptamer signal 82 nt upstream of the stop codon of ORF1 suggests that a -1 translational frameshifting strategy is involved in the expression of ORF2, like in other totiviruses. Phylogenetic analysis based on the CP and RdRP amino acid sequences placed this virus within a clade of plant-associated totiviruses, with taro-associated virus as its closest relative. Hence, based on its distinct host and the amino acid sequence similarity between BTLV and its relatives, we conclude that birch toti-like virus is a new member of the genus Totivirus.

Genomic characterization of an Omono River virus isolated from Culex tritaeniorhynchus in eastern China.

Omono River virus (OMRV) is a newly reported, unclassified RNA virus in the family Totiviridae, which infects mosquitoes and bats. In this study, we report the isolation of an OMRV strain SD76 from Culex tritaeniorhynchus captured in Jinan city, China. The cytopathic effect was characterized by cell fusion on C6/36 cell line. Its complete genome was 7611 nucleotides in length, with 71.4-90.4% similarities with other OMRV strains. Phylogenetic analysis based on complete genomes showed all OMRV-like strains can be divided into 3 groups with between-group distances ranging from 0.254 to 0.293. These results revealed that the OMRV isolate had high genetic diversity with those identified previously, and enriched the genetic information of family Totiviridae.

Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

Identification and molecular characterization of a novel member of the genus Totivirus from Areca catechu L.

In previous work, RNA-seq was applied to identify the causal agent of yellow leaf disease (YLD) in areca palm (Areca catechu L.), resulting in the identification of areca palm velarivirus 1 (APV1) associated with YLD. Additionally, RNA-seq revealed a totivirus-like virus in areca palm. This work revealed that the totivirus-like virus is prevalent in asymptomatic areca palms. Therefore, it was tentatively named "areca palm latent totivirus 1" (APLTV1). The complete genome sequence of APLTV1 was determined and found to be 4754 base pairs (bp) in length, containing two ORFs whose encoded proteins share 55% and 69% amino acid (aa) sequence identity with the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), respectively, of Bursera graveolens-associated totivirus 1 (BgAT1). Phylogenetic analysis based on alignment of the CP and RdRp sequences revealed that APLTV1 clustered with other members of the genus Totivirus, suggesting that APLTV1 represents a novel species of the genus Totivirus, family Totiviridae.

Molecular characterization of a novel victorivirus isolated from the phytopathogenic fungus Phaeobotryon rhois.

Phaeobotryon rhois is an important pathogenic fungus that causes dieback and canker disease of woody hosts. A novel mycovirus, tentatively named "Phaeobotryon rhois victorivirus 1" (PrVV1), was identified in P. rhois strain SX8-4. The PrVV1 has a double-stranded RNA (dsRNA) genome that is 5,224 base pairs long and contains two open reading frames (ORF1 and ORF2), which overlap at a AUGA sequence. ORF1 encodes a polypeptide of 786 amino acids (aa) that contains the conserved coat protein (CP) domain of victoriviruses, while ORF2, encodes a large polypeptide of 826 aa that contains the conserved RNA-dependent RNA polymerase (RdRp) domain of victoriviruses. Our analysis of genomic structure, homology, and phylogeny indicated that PrVV1 is a novel member of the genus Victorivirus in the family Totiviridae. This is the first report of the complete genome sequence of a victorivirus that infects P. rhois.

Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

Potential origins of fish toti-like viruses in invertebrates.

The virus family Totiviridae had originally been considered to include only viruses which infected fungal and protist hosts, but since 2006 a growing number of viruses found in invertebrates and fish have been shown to cluster phylogenetically within this family. These Totiviridae-like, or toti-like, viruses do not appear to belong within any existing genera of Totiviridae, and whilst a number of new genus names have been suggested, none has yet been universally accepted. Within this growing number of toti-like viruses from animal hosts, there exists emerging viral threats particularly to aquaculture, namely Infectious myonecrosis virus in whiteleg shrimp and Piscine myocarditis virus (PMCV) in Atlantic salmon (Salmo salar). PMCV in particular continues to be an issue in salmon aquaculture as a number of questions remain unanswered about how the virus is transmitted and the route of entry into host fish. Using a phylogenetic approach, this study shows how PMCV and the other fish toti-like viruses probably have deeper origins in an arthropod host. Based on this, it is hypothesized that sea lice could be acting as a vector for PMCV, as seen with other RNA viruses in Atlantic salmon aquaculture and in the toti-like Cucurbit yellows-associated virus which is spread by the greenhouse whitefly Trialeurodes vaporariorum.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

A novel victorivirus isolated from the tobacco spot blight fungus Stagonosporopsis cucurbitacearum in China.

Stagonosporopsis cucurbitacearum is an important plant-pathogenic fungus that causes stem and leaf blight diseases in a variety of crops. Here, we report the characterization of a novel victorivirus, tentatively named "Stagonosporopsis cucurbitacearum victorivirus 1" (ScVV-1), isolated from the S. cucurbitacearum isolate M-7. The ScVV-1 genome is 5,165 bp in length with a predicted GC content of 60.1% and contains two large open reading frames (ORF 1 and ORF2) encoding putative proteins that share significant sequence similarity with coat proteins (CPs) and RNA-dependent RNA polymerases (RdRps) of mycoviruses of the family Totiviridae. The ScVV-1 RdRp appears to be translated using a stop-initiation pentanucleotide UAAUG sequence. Phylogenetic analysis based on CP and RdRp amino acid (aa) sequences both indicated that ScVV-1 belongs to the genus Victorivirus in the family Totiviridae. To our knowledge, this is the first full-length genome sequence of a victorivirus infecting S. cucurbitacearum.

Identification of novel totiviruses from the ascomycetous fungus Geotrichum candidum.

Mycoviruses are widely distributed across the kingdom Fungi, including ascomycetous yeast strains of the class Saccharomycetes. Geotrichum candidum is an important fungal pathogen belonging to Saccharomycetes and has a diverse host range. Here, we report the characterization of four new classical totiviruses from two distinct Geotrichum candidum strains from Pakistan. The four identified viruses were tentatively named "Geotrichum candidum totivirus 1, 2, 3a, and 3b" (GcTV1-3b). The complete dsRNA genomes of the identified totiviruses are 4621, 4592, 4576, and 4576 bp in length, respectively. All totivirus genomes have two open reading frames, encoding a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP), respectively. The downstream RdRP domain is assumed to be expressed as a CP-RdRP fusion product via -1 frameshifting mediated by a heptameric slippery site. Sequence comparisons and phylogenetic analysis showed that each of the discovered viruses belongs to a new species of the genus Totivirus in the family Totiviridae, with GcTV1 and GcTV3 (a and b strains) clustering in one subgroup and GcTV2 in another subgroup.

Complete genome sequence of a novel victorivirus infecting cicada flower (Cordyceps chanhua).

Cicada flower, Cordyceps chanhua, is a precious edible and medicinal mushroom with uses in both medicine and food in China. In this study, Cordyceps chanhua strain RCEF5995 was found to be coinfected by a previously characterized alternavirus, Cordyceps chanhua alternavirus 1 (CcAV1), and a novel victorivirus, tentatively named "Cordyceps chanhua victorivirus 1" (CcV1). Molecular characterization of CcV1 showed that its complete genome is 5,232 nucleotides long with a GC content of 57.5%. Sequence analysis indicated that CcV1 contains two overlapping open reading frames (ORFs), ORF1 and ORF2, encoding a putative coat protein (CP) of 742 amino acids (aa) and a putative RNA-dependent RNA polymerase (RdRp) of 836 aa, respectively. The termination codon of the CP ORF overlaps with the initiation codon of the RdRp ORF at the tetranucleotide sequence AUGA. Homolog searches, sequence comparisons, and phylogenetic analysis based on deduced amino acid sequences of RdRp indicated that CcV1 is a new member of the genus Victorivirus, family Totiviridae.

Molecular characterization of a novel victorivirus infecting Corynespora cassiicola.

A novel victorivirus was detected in an isolate of Corynespora cassiicola strain 20180909-03 and was named "Corynespora cassiicola victorivirus 1" (CcVV1). The complete genome sequence of this virus is 5140 bp in length and contains 57% GC with two large open reading frames (ORFs) overlapping at the tetranucleotide AUGA. The ORFs were predicted to encode a coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), respectively, which are conserved in dsRNA fungal viruses of the family Totiviridae. Comparison and phylogenetic analysis of the deduced amino acid sequences of RdRp and CP showed that CcVV1 is a new member of the genus Victorivirus. This is the first report of a genomic sequence of a victorivirus infecting Corynespora cassiicola.

Complete genome sequence of a novel victorivirus infecting Aspergillus niger.

Aspergillus niger is an important filamentous phytopathogenic fungus with a broad host range. A novel double-stranded (ds) RNA mycovirus, named Aspergillus niger victorivirus 1 (AnV1), isolated from A. niger strain baiyun3.23-4, was sequenced and analyzed. The AnV1 genome is 5317 nucleotides long with a GC content of 56%. AnV1 contains two open reading frames (ORF1 and 2), overlapping at a tetranucleotide sequence (AUGA). ORF1 encodes a putative capsid protein (CP) of 778 amino acids (aa), while ORF2 potentially encodes a putative RNA-dependent RNA polymerase (RdRp) of 826 aa. Phylogenetic analysis indicated that AnV1 is a new member of the genus Victorivirus in the family Totiviridae. As far as we know, this is the first report of the complete genome sequence of a victorivirus infecting A. niger.

Molecular characterization of a novel victorivirus isolated from Botryosphaeria dothidea, the causal agent of longan leaf spot disease.

Mycoviruses are widespread in all major taxonomic groups of filamentous fungi. Previous research has indicated that mycoviruses are associated with the phytopathogenic fungus Botryosphaeria dothidea. In this study, three distinct double-stranded RNA viruses were detected in B. dothidea strain YCLYY11 isolated from a leaf spot of longan (Dimocarpus longana). The results of BLAST analysis revealed that the predicted amino acid sequences of those viruses were similar to those of Botryosphaeria dothidea chrysovirus 1, Botryosphaeria dothidea partitivirus 1, and an apparent novel victorivirus. Sequencing and analysis of the complete genome of the novel victorivirus indicated it is 5218 bp in length and contains two open reading frames (ORFs) that overlap at the tetranucleotide AUGA. BLASTp analysis of the proteins encoded by ORF1 and ORF2 showed that they were most similar to the coat protein and RNA-dependent RNA polymerase of Sphaeropsis sapinea RNA virus 2 (81.37% and 74.09% identical, respectively). A phylogenetic tree showed that the novel virus clustered together with victoriviruses and was separate from members of the other four genera of the family Totiviridae. Based on its genome structure and the results of phylogenetic analysis, we propose that this novel victorivirus should be named "Botryosphaeria dothidea victorivirus 3". This is also the first report of these three mycoviruses coinfecting a strain of B. dothidea.

Characterization of a novel victorivirus from Nigrospora chinensis, a fungus isolated from tobacco.

Here, we characterized a new mycovirus from the fungus Nigrospora chinensis, which was named "Nigrospora chinensis victorivirus 1" (NcVV1). The NcVV1 genome is 5283 bp in length, containing two continuous open reading frames (ORFs), ORF1 and ORF2. ORF1 and ORF2 were predicted to encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), respectively. The stop codon of ORF1 overlaps with the start codon of ORF2 by the tetranucleotide sequence AUGA. Phylogenetic analysis based on amino acid sequences of RdRp and CP indicated that NcVV1 clustered with members of the genus Victorivirus in the family Totiviridae. To our knowledge, this was the first report of a mycovirus infecting N. chinensis.

Characterization of early phases of cardiomyopathy syndrome pathogenesis in Atlantic salmon (Salmo salar L.) through various diagnostic methods.

Since the first description of cardiomyopathy syndrome (CMS) in Atlantic salmon, in 1985, the disease caused by piscine myocarditisvirus (PMCV) has become a common problem in Atlantic salmon farming, not only in Norway, but also in other salmon farming countries like Scotland and Ireland. In the last years, CMS has been ranked as the most important salmon viral disease in Norway regarding both mortality and economic losses. Detailed knowledge of infection and pathogenesis is still lacking, a decade after the causal agent was first described, and there is a need for a wider range of methods/tools for diagnostic and research purposes. In this study, we compared the detection of PMCV- and CMS-related tissue lesions using previously used and well-known methods like histopathology and real-time RT-PCR to immunohistochemistry (IHC), a less used method, and a new method, RNAscope in situ hybridization. Tissue samples of three different cardiac compartments, mid-kidney and skin/muscle tissue were compared with non-lethal parallel samplings of blood and mucus. The development of pathological cardiac lesions observed in this experiment was in accordance with previous descriptions of CMS. Our results indicate a viremic phase 10- to 20-day post-challenge (dpc) preceding the cardiac lesions. In this early phase, virus could also be detected in relatively high amount in mid-kidney by real-time RT-PCR. Plasma and/or mid-kidney samples may, therefore, be candidates to screen for early-phase PMCV infection. The RNAscope in situ hybridization method showed higher sensitivity and robustness compared with the immunohistochemistry and may be a valuable support to histopathology in CMS diagnostics, especially in cases of untypical lesions or mixed infections.

Metatranscriptomic Analysis Reveals Rich Mycoviral Diversity in Three Major Fungal Pathogens of Rice.

In recent years, three major fungal diseases of rice, i.e., rice blast, rice false smut, and rice-sheath blight, have caused serious worldwide rice-yield reductions and are threatening global food security. Mycoviruses are ubiquitous in almost all major groups of filamentous fungi, oomycetes, and yeasts. To reveal the mycoviral diversity in three major fungal pathogens of rice, we performed a metatranscriptomic analysis of 343 strains, representing the three major fungal pathogens of rice, Pyricularia oryzae, Ustilaginoidea virens, and Rhizoctonia solani, sampled in southern China. The analysis identified 682 contigs representing the partial or complete genomes of 68 mycoviruses, with 42 described for the first time. These mycoviruses showed affinity with eight distinct lineages: Botourmiaviridae, Partitiviridae, Totiviridae, Chrysoviridae, Hypoviridae, Mitoviridae, Narnaviridae, and Polymycoviridae. More than half (36/68, 52.9%) of the viral sequences were predicted to be members of the families Narnaviridae and Botourmiaviridae. The members of the family Polymycoviridae were also identified for the first time in the three major fungal pathogens of rice. These findings are of great significance for understanding the diversity, origin, and evolution of, as well as the relationship between, genome structures and functions of mycoviruses in three major fungal pathogens of rice.

Characterisation of Trichomonas vaginalis Isolates Collected from Patients in Vienna between 2019 and 2021.

Trichomonas vaginalis (TV) is the causative agent of trichomoniasis, the most common nonviral sexually transmitted disease. TV can carry symbionts such as Trichomonas vaginalis virus (TVV) or Mycoplasma hominis. Four distinct strains of TV are known: TVV1, TVV2, TVV3, and TVV4. The aim of the current study was to characterise TV isolates from Austrian patients for the presence of symbionts, and to determine their effect on metronidazole susceptibility and cytotoxicity against HeLa cells. We collected 82 TV isolates and detected presence of TVV (TVV1, TVV2, or TVV3) in 29 of them (35%); no TVV4 was detected. M. hominis was detected in vaginal/urethral swabs by culture in 37% of the TV-positive patients; M. hominis DNA was found in 28% of the TV isolates by PCR. In 15% of the patients, M. hominis was detected in the clinical samples as well as within the respective TV isolates. In 22% of the patients, M. hominis was detected by culture only. In 11 patients, M. hominis was detected only within the respective cultured TV isolates (13%), while the swab samples were negative for M. hominis. Our results provide a first insight into the distribution of symbionts in TV isolates from Austrian patients. We did not observe significant effects of the symbionts on metronidazole susceptibility, cytotoxicity, or severity of symptoms.